Acoustic Ejection Native Mass Spectrometry for High-Throughput Protein-Ligand Screening

Area of Science:

• Biochemistry
• Analytical Chemistry
• Structural Biology

Background:

• Native mass spectrometry (nMS) analyzes protein-ligand interactions, but throughput limitations hinder library screening.
• nMS provides insights into binding affinity, stoichiometry, and protein oligomeric states.
• Drug discovery requires efficient methods for characterizing non-covalent protein-ligand interactions.

Purpose of the Study:

• To develop and evaluate a high-throughput platform for native mass spectrometry screening using acoustic ejection mass spectrometry (AEMS).
• To assess the utility of AEMS-nMS for identifying non-covalent protein-ligand binders and monitoring protein oligomeric states.
• To demonstrate the speed and reproducibility of AEMS for screening compound libraries.

Main Methods:

• Coupling acoustic ejection mass spectrometry (AEMS) to a high-resolution QTOF mass spectrometer for nMS.
• Utilizing SARS-CoV-2 main protease (Mpro) as a model system to evaluate AEMS-nMS.
• Screening a 1536-well plate with rapid acquisition times per well.

Main Results:

• AEMS-nMS successfully identified non-covalent binders and compounds affecting the Mpro monomer-dimer equilibrium.
• Simultaneous assessment of ligand binding and protein oligomeric state was achieved.
• Screening of a 1536-well plate was completed in approximately 1 hour with excellent reproducibility.

Conclusions:

• Acoustic ejection nMS is a practical high-throughput screening platform for analyzing protein-ligand interactions.
• This method provides direct molecular information on binding, stoichiometry, and oligomeric state.
• AEMS-nMS significantly enhances the throughput of native mass spectrometry for drug discovery applications.